The present invention relates to immunoaffinity chromatography systems, and particularly to immunoaffinity processes employing a monoclonal antibody having divalent cation-dependent affinity for a selected peptide determinant.
The prior art describes recombinant fusion proteins which comprise synthetic leader peptides or protein fragments linked to independently derived polypeptides. In such fusions, the leader peptide or protein fragment can facilitate protein expression and purification by providing, for example, enzymatic activity enabling identification of recombinants, an amino acid sequence recognized by cellular secretory mechanisms, or a sequence having distinctive chemical or antigenic characteristics useful in purifying the fusion protein by ion exchange, reverse phase, or affinity chromatographic media.
Itakura, U.S. Pat. No. 4,571,421, describes hybrid polypeptides consisting of a somatostatin sequence and a fragment of .beta.-galactosidase enzyme, separated by a CnBr-cleavable site permitting separation of the two protein segments. In this system, the presence of the .beta.-galactosidase fragment permits identification of recombinants bearing the somatostatin sequence. Schuman et al., J. Biol. Chem. 255:168 (1980), and Reed et al., Gene 20:255 (1982) disclose variations of this approach involving fusion of nucleotide sequences encoding biologically active fragments of .beta.-galactosidase and newly isolated genes. The translated hybrid protein was isolated by reference to the physical and enzymatic properties of .beta.-galactosidase, and used to prepare specific antisera to the product of the newly isolated genes.
Rutter, Published European Patent Application No. 35384 (1981), discloses DNA constructions used to facilitate expression of cloned DNA sequences. Among the constructions disclosed are sequences encoding fusion proteins comprising an N-terminal sequence having distinctive physical properties useful for purification, joined to a desired C-terminal portion via a sequence which can be specifically cleaved to remove the N-terminal sequence. An example of such a cleavage sequence is the peptide sequence DDDDK recognized by enterokinase. Sequences having particular properties useful for purification include polyanionic segments and polycationic segments that will bind readily to ion exchangers, and hydrophobic segments capable of binding to reverse-phase media. This reference also discloses hybrid fusion proteins comprising fragments capable of being bound by specific antibody in an affinity chromatography step.
Brewer et al., U.S. Pat. No. 4,532,207, disclose recombinant fusion proteins comprising a charged polymer of amino acids, for example, polyarginine, linked to a polypeptide of interest. Following expression in a microbial host, the fusion protein is purified by chromatography involving binding of the charged polymer to ion-exchange media. Following purification, the charged polymer is removed by controlled digestion with an exopeptidase. Smith et al., Gene 32:321 (1984) and Sassenfeld, Bio/Technology, January 1984, p. 76, also describe aspects of this approach to recombinant protein purification.
Improvements in recombinant protein expression and purification technologies are of considerable interest to the biotechnology, pharmaceutical, and chemical industries.